# US 11,306,333 B2 · Wood-Ljungdahl microorganisms that produce polyhydroxybutyrate

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- Number: US 11,306,333 B2
- Document ID: US11306333B2
- Status: Granted
- Kind: B2
- Application number: 16/152,354
- Filed: 2018-10-04
- Published: 2019-04-25
- Granted: 2022-04-19
- Earliest claimed: 2017-10-04
- Assignees: LanzaTech, Inc.
- Applicants: LanzaTech, Inc.
- Inventors: Ryan Christopher Tappel; James Bruce Yarnton Haycock Behrendorff; Michael Koepke; Esteban Marcellin; Renato de Souza Pinto Lemgruber; Kaspar Valgepea; Lars Nielsen
- Web page: https://patentgrep.com/patent/US11306333B2

## CPC

- C12P 7/625: Polyesters of hydroxy carboxylic acids
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12Y 101/01036: Acetoacetyl-CoA reductase (1.1.1.36)
- C12Y 101/01157: 3-Hydroxybutyryl-CoA dehydrogenase (1.1.1.157)
- C12Y 203/01: transferring groups other than amino-acyl groups (2.3.1)
- C12Y 203/01009: Acetyl-CoA C-acetyltransferase (2.3.1.9)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The invention provides microorganisms and methods for the production of polyhydroxybutyrate (PHB) from gaseous substrates. In particular, the invention provides a non-naturally occurring Wood-Ljungdahl microorganism comprising (a) an enzyme that converts acetyl-CoA to acetoacetyl-CoA, (b) an enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and (c) an enzyme that converts 3-hydroxybutyryl-CoA to polyhydroxybutyrate, and methods related thereto.

## Claims

### Claim 1

A non-naturally occurring Wood-Ljungdahl microorganism capable of producing polyhydroxybutyrate comprising:
a. a nucleic acid encoding a heterologous acetyl-CoA C-acetyltransferase (EC 2.3.1.9) enzyme that converts acetyl-CoA to acetoacetyl-CoA,
b. a nucleic acid encoding a heterologous acetoacetyl-CoA reductase (EC 1.1.1.36) or a nucleic acid encoding a heterologous 3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.157) enzyme that converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and
c. a nucleic acid encoding a heterologous polyhydroxyalkanoate synthase (EC 2.3.1.-) enzyme that converts 3-hydroxybutyryl-CoA to polyhydroxybutyrate.

### Claim 2 (depends on 1)

The microorganism of claim 1, wherein the acetyl-CoA C-acetyltransferase is derived from a parental microorganism selected from the group consisting of Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Clostridium acetobutylicum, Cupriavidus necator, Escherichia coli, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida, and Streptomyces coelicolor.

### Claim 3 (depends on 1)

The microorganism of claim 1, wherein the acetoacetyl-CoA reductase is derived from a parental microorganism selected from the group consisting of Acinetobacter baumannii, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida, and Streptomyces coelicolor.

### Claim 4 (depends on 1)

The microorganism of claim 1, wherein the 3-hydroxybutyryl-CoA dehydrogenase is derived from Clostridium beijerinckii, Clostridium acetobutylicum, or Clostridium kluyveri.

### Claim 5 (depends on 1)

The microorganism of claim 1, wherein the polyhydroxyalkanoate synthase is derived from a parental microorganism selected from the group consisting of Acinetobacter baumannii, Aeromonas caviae, Aeromonas hydrophilia, Alcaligenes latus, Arthrospira platensis, Bacillus subtilis, Burkholderia cepacia, Cupriavidus necator, Haloferax mediterranei, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas mandelii, Pseudomonas oleovorans, Pseudomonas putida, Pseudomonas sp. 61-3, Rhodospirillum rubrum, and Streptomyces coelicolor.

### Claim 6 (depends on 1)

The microorganism of claim 1, wherein the microorganism is a member of a genus selected from the group consisting of Acetobacterium, Alkalibaculum, Blautia, Butyribacterium, Clostridium, Eubacterium, Moorella, Oxobacter, Sporomusa, and Thermoanaerobacter.

### Claim 7 (depends on 1)

The microorganism of claim 1, wherein the microorganism is derived from a parental microorganism selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.

### Claim 8 (depends on 7)

The microorganism of claim 7, wherein the microorganism is derived from a parental bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium coskatii, Clostridium ljungdahlii, and Clostridium ragsdalei.

### Claim 9 (depends on 1)

The microorganism of claim 1, wherein the microorganism consumes gaseous substrates comprising one or more of CO, CO2, and H2.

### Claim 10 (depends on 1)

The microorganism of claim 1, wherein the microorganism is anaerobic.

### Claim 11 (depends on 1)

The microorganism of claim 1, wherein the microorganism is not capable of degrading polyhydroxybutyrate.

### Claim 12 (depends on 1)

The microorganism of claim 1, wherein the microorganism is not phototrophic, photosynthetic, or methanotrophic.

### Claim 13 (depends on 1)

A method of producing polyhydroxybutyrate comprising culturing the microorganism of claim 1 in the presence of a gaseous substrate, whereby the microorganism produces polyhydroxybutyrate.

### Claim 14 (depends on 13)

The method of claim 13, wherein the gaseous substrate comprises one or more of CO, CO2, and H2.

### Claim 15 (depends on 13)

The method of claim 13, wherein the culturing is performed under anaerobic conditions.

### Claim 16 (depends on 13)

The method of claim 13, wherein the culturing is performed in the absence of carbohydrate substrates.

### Claim 17 (depends on 13)

The method of claim 13, wherein the culturing is performed in the absence of light.